Infections caused by mycobacteria are responsible for severe morbidity and mortality. Mycobacterium tuberculosis and Mycobacterium avium are intracellular pathogens that infect both healthy individuals and immunocompromised patients. M. avium are usually resistant to conventional anti-tuberculosis therapy, and with the few drugs shown to have anti-M. avium activity in humans, such as the new macrolides, treatment or prolonged prophylaxis of disseminated disease selects resistant mutants after a course of monotherapy. In addition, multiple outbreaks of multi-drug resistant M. tuberculosis have created clinical challenges for hospital and community management of patients. The goal of this proposal is to be focused and apply new strategies to identify and characterize the targets of mefloquine, a drug just recognized to have activity against mycobacteria. We have found that mefloquine has in vitro activity against both M. avium and M. tuberculosis and is borderline bactericidal against M. avium organisms in mice (we have not tested the activity in vivo against M. tuberculosis). Because mefloquine can achieve tissue concentrations 80-fold greater than serum levels, and mycobacteria survive intracellularly and have a long half-life, this class of compound has potential to become part of anti-mycobacterial regimen. Furthermore, mefloquine is active against M. avium strain resistant to macrolides, quinolones, isoniazid, ethambutol and rifampin, suggesting a novel mechanism of action. Therefore, we believe that determining the biochemical target of mefloquine in mycobacteria can lead the way to developing compounds with even more potent activity. The results thus far obtained with mefloquine suggest that it is the first very active drug identified against mycobacteria in years. Specifically, we plan to: (1) clone the mefloquine resistant-determinant using resistant mutants. In addition, by using a M. avium promoter library cloned in a reporter construct (green fluorescent protein), we plan to determine the pathways in the bacterium that are inhibited or stimulated when M. avium is exposed to mefloquine. This work, focused on an active anti-mycobacterial compound, has the potential to unveil new target(s) in both M. avium and M. tuberculosis.